Computational protocol: Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena)

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Protocol publication

[…] Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most harmful plant-pathogenic bacteria worldwide (), causing tremendous yield losses within Solanaceae and Musaceae (, ). The R. solanacearum species complex (RSSC) is composed of four phylotypes (I, II, III, and IV). Among these, the pandemic phylotype I displays population structure (demographic expansion) and genomic features (recombination traces) probably at the origin of its high evolutionary potential (), allowing it to adapt to new host species or resistant accessions of a host species. Among Solanaceae, eggplant is the crop species that carries the highest levels of resistance to R. solanacearum. So far, AG91-25 is the first eggplant genotype whose bacterial wilt resistance has been genetically mapped and associated with one major resistance gene (ers1) (). Some R. solanacearum strains are totally controlled by AG91-25, while other strains are able to overcome its resistance (, ). To investigate the molecular bases driving these virulence differences, we sequenced nine strains of R. solanacearum that displayed different virulence patterns on AG91-25 (virulent, avirulent, and provoking latent infection) (J. Guinard, unpublished data; and ), paying particular attention to differences in the type III effector gene (T3E) content between strains. Of these nine strains, eight are of phylotype I and originate from Asia (RUN0969 and RUN0157), Africa (RUN0215 and RUN1744), the Indian Ocean (RUN3012, RUN3013, and RUN3014), and South America (RUN1985), whereas RUN0039 belongs to phylotype III and originates from West Africa.Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega). The strains were sequenced to a depth of ~100× using the Illumina HiSeq 2500 technology (150-base paired-end reads with an average insert size of 450 bp). The reads were quality trimmed using HTQC () before being assembled using CLC Genomics Workbench version 7.0.3 (CLC bio, Aarhus, Denmark). Genome assemblies were annotated automatically using the Web interface of the MicroScope platform (). The nine genomes consist of 266 to 728 contigs. The genome sizes of the phylotype I strains range from 5.57 to 5.84 Mb, while the phylotype III genome is shorter, at 5.42 Mb. The overall G+C contents range from 66.86 to 67.05%. The draft genomes contain between 5,198 and 5,426 coding sequences, with 48 to 52 tRNA genes and 2 to 3 complete rRNA loci.The genomes were submitted to the Ralstonia T3E website (https://iant.toulouse.inra.fr/bacteria/annotation/site/prj/T3Ev2/) () for T3E identification and rip-based annotation. The phylotype I strains contain 67 to 73 effector genes and two to seven pseudogenes, which are in the range of those in the reference phylotype I genome GMI1000 (). Conversely, the phylotype III RUN39 contains only 55 T3Es and seven pseudogenes, which constitutes a T3E repertoire smaller than that of the phylotype I and phylotype III reference strain CMR15 (). We did not find a clear association between T3E repertoire and virulence on AG91-25. However, the prevalence of the genes ripAX2, ripA1, ripC2, ripE2, ripS6, and ripP2 was different between avirulent and virulent strains. […]

Pipeline specifications

Software tools HTQC, CLC Genomics Workbench
Application Phylogenetics
Organisms Solanum melongena